Leptin is a neurohormone that acts in the hypothalamus to regulate energy balance and food intake (M. Wauters, et al., Eur. J. Endocrinol., 2000, 143, 293-311). Recessive mutations in the leptin or the gene for its receptor, ObR, result in profound obesity and type II diabetes mellitus (Y. Zhang, et al., Nature, 1994, 372, 425-432). In addition to its role as a neurohormone and energy regulator, leptin can modulate several physiological processes, such as fertility, lactation, immune response, bone remodeling, hematopoiesis, and cognitive functions. On a cellular level, leptin can act as a mitogen, survival factor, metabolic regulator, or pro-angiogenic factor (M. Wauters, et al., Eur. J. Endocrinol., 2000, 143, 293-311).
Mature human leptin is secreted as a 146-amino acid protein, with a bundle of 4 helices (helices A-D) with an up-up-down-down topology (F. Zhang, et al., Nature, 1997, 387, 206-209). Superimposition of the leptin sequence with other cytokines, such as human IL-6, bovine G-CSF, human oncostatin M, etc., reveals three potential bivalent receptor binding sites (sites mostly around the pairwise helices (F. Peelman et al., J. Biol. Chem., 2004, 279, 41038-41046).
Leptin binds to the extracellular domain of its receptor, ObR, which can be expressed as multiple isoforms. The long isoform of ObR (ObR1) can induce multiple intracellular signaling pathways, for instance, the classic cytokine JAK2/STAT3 (Janus kinase 2/signal transducer and activator of transcription 3) pathway; the Ras/ERK1/2 (Ras/extracellular signal-regulated kinases 1/2) signaling cascade; and the PI-3K/Akt/GSK3 (phosphoinositide 3 kinase/protein kinase B/glycogen synthase kinase 3) growth/antiapoptotic pathway. In addition, leptin has been found to induce PLC (phospholipase C)-γ, PKC (protein kinase C), p38 kinase, and nitric oxide (NO) production (C. Bjorbaek, et al., J. Biol. Chem., 1997, 272, 32686-32695; G. Sweeney, Cell. Signal., 2002, 14, 655-663; L. Zabeau, et al., FEBS. Lett., 2003, 546, 45-50). Ultimately, induction of ObR1 can activate several genes involved in cell proliferation, including c-fos, c-jun, junB, egr-1, and socs3, and upregulate the expression angiogenic factors, such as VEGF (G. Sweeney, Cell. Signal., 2002, 14, 655-663; L. Zabeau, et al., FEBS. Lett., 2003, 546, 45-50; K. A. Frankenberry, et al., Am. J. Surg., 2004, 188, 560-565).
The receptor binding site around residue 40 of leptin is labeled as site I. The residues at the very N-terminus and in the middle of the protein are labeled as binding site II, and the residues at the C-terminus as binding site III. Interfering with these binding surfaces may increase or decrease the efficiency of leptin/ObR binding and modulate downstream ObR signaling. Full-length leptin and point mutants of full-length leptin have been examined as potential therapeutic agents for obesity. However, results were disappointing, largely due to leptin resistance in obese people as well as difficulties in recombinant leptin delivery to the central nervous system (J. M. Montez, et al., Proc. Natl. Acad. Sci. USA, 2005, 102, 2537-2542).
As a first indication of the possibility of growth arrest upon ObR inactivation, the proliferation rate of leptin-sensitive BAF/3 cells stably transfected with the long form of human leptin receptor was measured after treatment of leptin fragments and their mutants (L. Niv-Spector et al., Biochem. J., 2005, 391, 221-230). It was found that single-point mutations in leptin binding site III lower the affinity between the ligand and the receptor, attenuating the agonistic activity and converting those mutants into both partial antagonists and weak agonists.
The multiple roles leptin plays in various biological processes suggest that it is not straightforward to obtain true leptin agonists or antagonists that do not change the downstream signaling effect upon varying environmental conditions. Indeed, the emergence of both partial antagonists and weak agonists as listed above indicates that, depending upon the cell lines used, as well as the presence or absence of native, unmodified leptin, the same mutant protein or large subunit can trigger different biological responses. The use of such proteins and peptides in human or veterinary therapy will therefore likely meet regulatory opposition, as the peptides do not demonstrate pure, controllable agonist activity of the leptin receptor.
What is needed is a leptin-based peptide agonist for use in leptin-deficient diseases such as obesity, lipodystrophy, diet induced food craving and anorexia-related infertility. The present invention addresses and meets these needs.